Humanized anti-CD3 specific antibodies

ABSTRACT

Novel aglycosylated antibodies having a binding affinity for the CD3 antigen complex are of value for use in therapy, particularly in immunosuppression.

This invention relates to novel antibodies, in particular to antibodiesdirected against the CD3 antigen complex.

Antibodies, or immunoglobulins, comprise two heavy chains linkedtogether by disulphide bonds and two light chains, each light chainbeing linked to a respective heavy chain by disulphide bonds in a “Y”shaped configuration. The two “arms” of the antibody are responsible forantigen binding, and include regions where the polypeptide structurevaries, these “arms” being termed Fab′ fragments(fragment-antigen-binding) or F(ab′)₂ which represents two Fab′ armslinked together by disulphide bonds. The “tail” or central axis of theantibody contains a fixed or constant sequence of peptides and is termedthe Fc fragment (fragment-crystalline). The production of monoclonalantibodies was first disclosed by Kohler and Milstein (Kohler &Milstein, Nature, 256, 495-497 (1975)). Such monoclonal antibodies havefound widespread use as diagnostic agents and also in therapy.

Each heavy chain has at one end a variable domain followed by a numberof constant domains. Each light chain has a variable domain at one endand a constant domain at its other end, the light chain variable domainbeing aligned with the variable domain of the heavy chain and the lightchain constant domain being aligned with the first constant domain ofthe heavy chain (CH1). The constant domains in the light and heavychains are not involved directly in binding the antibody to antigen. Thelight chain constant domain and the CH1 domain of the heavy chainaccount for 50% of each Fab′ fragment.

The variable domains of each pair of light and heavy chains form theantigen binding site. The domains on the light and heavy chains have thesame general structure and each domain comprises four framework regions,whose sequences are relatively conserved, connected by threecomplementarity determining regions (CORs) (Kabat et al. Sequences ofProteins of Immunological Interest, U.S. Department of Health and HumanServices (1987)). The four framework regions largely adopt a beta-sheetconformation and the CDRs form loops connecting, and in some casesforming part of, the beta-sheet structure. The CDRs are held in closeproximity by the framework regions and, with the CDRs from the otherdomain, contribute to the formation of the antigen binding site.

The human CD3 antigen consists of a minimum of four invariantpolypeptide chains, which are non-covalently associated with the T-cellreceptors on the surface of T-cells, and is generally now referred to asthe CD3 antigen complex. It is intimately involved in the process ofT-cell activation in response to antigen recognition by the T-cellreceptors.

All CD3 monoclonal antibodies can be used to sensitise T-cells tosecondary proliferative stimuli such as IL1 (interleukin 1) and IL2(interleukin 2). In addition, certain CD3 monoclonal antibodies arethemselves mitogenic for T-cells. This property is isotype dependent andresults from the interaction of the CD3 antibody Fc domain with Fcreceptors on the surface of accessory cells.

Rodent CD3 antibodies have been used to influence immunological statusby suppressing, enhancing or re-directing T-cell responses to antigens.They therefore have considerable therapeutic potential in the human foruse as an immunosuppressive agent, for example for the treatment ofrejection episodes following the transplantation of renal, hepatic andcardiac allografts. However their value is compromised by two mainfactors. The first is the antiglobulin response evoked due to thexenogeneic nature of the antibody. The second is the “first dose”syndrome experienced by patients following the initial administration ofthe antibody. The symptoms, which range in severity from fever andchills to pulmonary edema, and which in rare cases can cause death, arecaused by the elevated levels of circulating cytokines associated withCD3-antibody induced T-cell activation. This phenomenon requires thecross-linking of the CD3 antigen on the surface of T-cells to accessorycells through Fc receptors; such proliferation does not occur withF(ab′)₂ fragments of CD3 antibodies.

The first problem can be addressed by re-shaping or “humanising” thevariable region genes of antibodies and expressing them in associationwith relevant human constant domain genes. This reduces the non-humancontent of the monoclonal antibody to such a low level that anantiglobulin response is unlikely. Such a reshaped antibody with abinding affinity for the CD3 antigen complex is described in UK PatentApplication No. 9121126.8 (published as GB 2249310A) and its equivalents(European Patent Application No. 91917169.4, Japanese Patent ApplicationNo. 516117/91 and U.S. patent application Ser. No. 07/862,543).

There remains however the problem of the first dose response when theseantibodies are used in therapy. Aglycosylation of antibodies has beendescribed to reduce their ability to bind to Fc receptors in vitro insome cases. However, it is not predictable that this will be true of allantibodies, particularly in vivo, and aglycosylation may result in theintroduction into the antibody of novel and unpredictable propertiesincluding novel Fc binding characteristics causing other undesirableeffects. It is also possible that other undesirable properties notassociated with Fc binding may be introduced to the antibody.

Moreover, it is of course of vital importance that aglycosylation is notaccompanied by the loss of certain desirable features of Fc binding inaddition to the loss of the undesirable features such as thoseattributable to the first dose response.

It has now been found, however, that it is possible to produceaglycosylated CD3 antibodies of the IgG subclass which surprisinglyretain their antigen binding specificity and immunosuppressiveproperties and yet do not induce T cell mitogenesis in vitro and inducea reduced level of cytokine release in vivo, whilst still maintainingsome Fc binding ability.

Accordingly, the invention provides an aglycosylated IgG antibody havinga binding affinity for the CD3 antigen complex.

The term aglycosylated is employed in its normal usage to indicate thatthe antibodies according to the invention are not glycosylated. Althoughthe present invention can be applied to antibodies having a bindingaffinity for a non-human CD3 antigen complex, for example various othermammalian CD3 antigens for veterinary use, the primary value of theinvention lies in aglycosylated antibodies having an affinity for thehuman CD3 antigen complex for use in the human and the followingdiscussion is particularly directed to that context.

Further discussion of CD3 antigens is to be found in the report of theFirst International Workshop and Conference on Human LeukocyteDifferentiation Antigens and description of various glycosylatedantibodies directed against the CD3 antigen is also to be found in thereports of this series of Workshops and Conferences, particularly theThird and Fourth, published by Oxford University Press. Specificexamples of such antibodies include those described by Van Ller et al.,Euro. J. Immunol., 1987, 17, 1599-1604, Alegre et al., J. Immunol.,1991, 140, 1184, and by Smith et al., ibid, 1986, 16, 478, the lastpublication relating to the IgG1 antibody UCHT1 and variants thereof.However, of particular interest as the basis for aglycosylatedantibodies according to the present invention are the CDRs contained inthe antibodies OKT3 and YTH 12.5.14.2. The antibody OKT3 is discussed inpublications such as Chatenaud et al., Transplantation, 1991, 51, 334and the New England Journal of Medicine paper, 1985, 313, 339, and alsoin European Patent No. 0 018 795 and U.S. Pat. No. 4,361,539. Theantibody YTH 12.5.14.2 (hereinafter referred to as YTH 12.5) isdiscussed in publications such as Clark et al., European J. Immunol.,1989, 19, 381-388 and reshaped YTH 12.5 antibodies are the subject of UKPatent Application No. 9121126.8 and its equivalents, this applicationdescribing in detail the CDRs present in this antibody.

Aglycosylated antibodies containing one or more of the CORs described inthe above application are of particular interest. Thus the antibodies ofthe invention preferably have at least one CDR selected from the aminoacid sequences:

-   (a) Ser-Phe-Pro-Met-Ala (SEQUENCE ID NO. 1),-   (b)    Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr-Arg-Asp-Ser-Val-Lys-Gly    (SEQUENCE ID NO. 2),-   (c) Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr (SEQUENCE ID NO. 3),-   (d) Thr-Leu-Ser-Ser-Gly-Asn-Ile-Glu-Asn-Asn-Tyr-Val-His (SEQUENCE ID    NO. 4),-   (e) Asp-Asp-Asp-Lys-Arg-Pro-Asp (SEQUENCE ID NO. 5),-   (f) His-Ser-Tyr-Val-Ser-Ser-Phe-Asn-Val (SEQUENCE ID NO. 6), and    conservatively modified variants thereof.

The term “conservatively modified variants” is one well known in the artand indicates variants containing changes which are substantiallywithout effect on antibody-antigen affinity.

The CDRs are situated within framework regions of the heavy chain (for(a), (b) and (c)) and light chain (for (d), (e) and (f)) variabledomains. The antibody also comprises a constant domain.

In a preferred embodiment the aglycosylated antibody has three CDRscorresponding to the amino acid sequences (a), (b) and (c) above orconservatively modified variants thereof and/or three CDRs correspondingto amino acid sequences (d), (e) and (f) or conservatively modifiedvariants thereof, the heavy chain CDRs (a), (b) and (c) being of mostimportance.

A preferred aglycosylated antibody with a binding affinity for the CD3antigen thus has a heavy chain with at least one CDR and particularlythree CDRs selected from the amino acid sequences:

-   (a) Ser-Phe-Pro-Met-Ala (SEQUENCE ID NO. 1),-   (b)    Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr-Arg-Asp-Ser-Val-Lys-Gly    (SEQUENCE ID NO. 2),-   (c) Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr (SEQUENCE ID NO. 3),    and conservatively modified variants thereof, and/or a light chain    with at least one CDR and particularly three CDRs selected from the    amino acid sequences:-   (d) Thr-Leu-Ser-Ser-Gly-Asn-Ile-Glu-Asn-Asn-Tyr-Val-His (SEQUENCE ID    NO. 4),-   (e) Asp-Asp-Asp-Lys-Arg-Pro-Asp (SEQUENCE ID NO. 5),-   (f) His-Ser-Tyr-Val-Ser-Ser-Phe-Asn-Val (SEQUENCE ID NO. 6),    and conservatively modified variants thereof.

Where an aglycosylated antibody according to the invention containspreferred CDRs as described hereinbefore it conveniently contains bothone or more of the specified heavy chain CDRs and one or more of thespecified light chain CDRs. The CDRs (a), (b) and (c) are arranged inthe heavy chain in the sequence: framework region 1/(a)/framework region2/(b)/framework region 3/(c)/framework region 4 in a leader→constantdomain (n-terminal to C-terminal) direction and the CDRs (d), (e) and(f) are arranged in the light chain in the sequence: framework region1/(d)/framework region 2/(e)/framework region 3/(f)/framework region 4in a leader→constant domain direction. It is preferred, therefore, thatwhere all three are present the heavy chain CDRs are arranged in thesequence (a), (b), (c) in a leader→constant domain direction and thelight chain CDRs are arranged in the sequence (d), (e), (f) in aleader→constant domain direction.

It should be appreciated however, that aglycosylated antibodiesaccording to the invention may contain quite different CDRs from thosedescribed hereinbefore and that, even when this is not the case, it maybe possible to have heavy chains and particularly light chainscontaining only one or two of the CDRs (a), (b) and (c) and (d), (e) and(f), respectively. However, although the presence of all six CDRsdefined above is therefore not necessarily required in an aglycosylatedantibody according to the present invention, all six CDRs will mostusually be present in the most preferred antibodies. A particularlypreferred aglycosylated antibody therefore has a heavy chain with threeCDRs comprising the amino acid sequences (a), (b) and (c) orconservatively modified variants thereof and a light chain with threeCDRs comprising the amino acid sequences (d), (e) and (f) orconservatively modified variants thereof in which the heavy chain CDRsare arranged in the order (a), (b), (c) in the leader constant regiondirection and the light chain CDRs are arranged in the order (d), (e),(f) in the leader constant region direction.

The CDRs may be of different origin to the variable framework regionand/or to the constant region and, since the CDRs will usually be of rator mouse origin, this is advantageous to avoid an antiglobulin responsein the human, although the invention does extend to antibodies with suchregions of rat or mouse origin.

More usually the CDRs are either of the same origin as the variableframework region but of a different origin from the constant region, forexample in a part human chimaeric antibody, or, more commonly, the CDRsare of different origin from the variable framework region.

The preferred CDRs discussed hereinbefore are obtained from a rat CD3antibody. Accordingly, although the variable domain framework region cantake various forms, it is conveniently of or derived from those of arodent, for example a rat or mouse, and more preferably of or derivedfrom those of human origin. One possibility is for the antibody to havea variable domain framework region corresponding to that in the YTH12.5hybridoma although the constant region will still preferably be of orderived from those of human origin. However the antibody of theinvention is preferably in the humanised form as regards both thevariable domain framework region and as discussed further hereinafter,the constant region.

Accordingly, the invention further comprises an aglycosylated antibodywhich has a binding affinity for the human CD3 antigen and in which thevariable domain framework regions and/or the constant region are of orare derived from those of human origin.

Certain human variable domain framework sequences will be preferable forthe grafting of the preferred CDR sequences, since the 3-dimensionalconformation of the CDRs will be better maintained in such sequences andthe antibody will retain a high level of binding affinity for theantigen. Desirable characteristics in such variable domain frameworksare the presence of key amino acids which maintain the structure of theCDR loops in order to ensure the affinity and specificity of theantibody for the CD3 antigen, the lambda type being preferred for thelight chain.

Human variable region frameworks which are particularly suitable for usein conjunction with the above CDRs have been previously identified in UKPatent Application No. 9121126.8. The heavy chain variable (V) regionframeworks are those coded for by the human VH type III gene VH26.D.J.which is from the B cell hybridoma cell line 18/2 (Genbank Code:Huminghat, Dersimonian et al., Journal of Immunology, 139, 2496-2501).The light chain variable region frameworks are those of the human V_(L)λtype VI gene SUT (Swissprot code; LV6CSHum, Solomon et al. In Glenner etal (Eds), Amyloidosis, Plenum Press N.Y., 1986, p. 449.

The one or more preferred CDRs of the heavy chain of the rat anti-CD3antibody are therefore preferably present in a human variable domainframework which has the following amino acid sequence reading in theleader→constant region direction, CDR indicating a CDR (a), (b) or (c)as defined hereinbefore, a conservatively modified variant thereof or analternativeCDR:—Glu-Val-Gln-Leu-Leu-Glu-Ser-Gly-Gly-Gly-Leu-Val-Gln-Pro-Gly-Gly-Ser-Leu-Arg-Leu-Ser-Cys-Ala-Ala-Ser-Gly-Phe-Thr-Phe-Ser-/CDR/-Trp-Val-Arg-Gln-Ala-Pro-Gly-Lys-Gly-Leu-Glu-Trp-Val-Ser-/CDR/-Arg-Phe-Thr-Ile-Ser-Arg-Asp-Asn-Ser-Lys-Asn-Thr-Leu-Tyr-Leu-Gln-Met-Asn-Ser-Leu-Arg-Ala-Glu-Asp-Thr-Ala-Val-Tyr-Tyr-Cys-Ala-Lys-/CDR/-Trp-Gly-Gln-Gly-Thr-Leu-Val-Thr-Val-Ser-Ser(SEQUENCE ID NO. 7/CDR/SEQUENCE ID NO. 8/CDR/SEQUENCE ID NO.9/CDR/SEQUENCE ID NO. 10).

In an aglycosylated antibody containing all three preferred CDRs, theheavy chain variable region comprises the following sequence:— (SEQUENCEID NO. 11) Glu-Val-Gln-Leu-Leu-Glu-Ser-Gly-Gly-Gly-Leu-Val-Gln-Pro-Gly-Gly-Ser-Leu-Arg-Leu-Ser-Cys-Ala-Ala-Ser-Gly-Phe-Thr-Phe-Ser-Ser-Phe-Pro-Met-Ala-Trp-Val-Arg-Gln-Ala-Pro-Gly-Lys-Gly-Leu-Glu-Trp-Val-Ser-Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr-Arg-Asp-Ser-Val-Lys-Gly-Arg-Phe-Thr-Ile-Ser-Arg-Asp-Asn-Ser-Lys-Asn-Thr-Leu-Tyr-Leu-Gln-Met-Asn-Ser-Leu-Arg-Ala-Glu-Asp-Thr-Ala-Val-Tyr-Tyr-Cys-Ala-Lys-Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr-Trp-Gly-Gln-Gly-Thr-Leu-Val-Thr-Val-Ser-Ser.

Similarly, the one or more preferred CDRs of the light chain of the ratCD3 antibody are therefore preferably present in a human variable domainframework which has the following amino acid sequence reading in theleader→constant region direction, CDR indicating a CDR (d), (e) and (f)as defined hereinbefore, a conservatively modified variant thereof or analternativeCDR:—Asp-Phe-Met-Leu-Thr-Gln-Pro-His-Ser-Val-Ser-Glu-Ser-Pro-Gly-Lys-Thr-Val-Ile-Ile-Ser-Cys-/CDR/-Trp-Tyr-Gln-Gln-Arg-Pro-Gly-Arg-Ala-Pro-Thr-Thr-Val-Ile-Phe-/CDR/-Gly-Val-Pro-Asp-Arg-Phe-Ser-Gly-Ser-Ile-Asp-Arg-Ser-Ser-Asn-Ser-Ala-Ser-Leu-Thr-Ile-Ser-Gly-Leu-Gln-Thr-Glu-Asp-Glu-Ala-Asp-Tyr-Tyr-Cys-/CDR/-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Thr-Val-Leu(SEQUENCE ID NO. 12/CDR/SEQUENCE ID NO. 13/CDR/SEQUENCE ID NO.14/CDR/SEQUENCE ID NO. 15).

In an aglycosylated antibody containing all three preferred CDRs thelight chain variable region comprises the following sequence:— (SEQUENCEID NO. 16) Asp-Phe-Met-Leu-Thr-Gln-Pro-His-Ser-Val-Ser-Glu-Ser-Pro-Gly-Lys-Thr-Val-Ile-Ile-Ser-Cys-Thr-Leu-Ser-Ser-Gly-Asn-Ile-Glu-Asn-Asn-Tyr-Val-His-Trp-Tyr-Gln-Gln-Arg-Pro-Gly-Arg-Ala-Pro-Thr-Thr-Val-Ile-Phe-Asp-Asp-Asp-Lys-Arg-Pro-Asp-Gly-Val-Pro-Asp-Arg-Phe-Ser-Gly-Ser-Ile-Asp-Arg-Ser-Ser-Asn-Ser-Ala-Ser-Leu-Thr-Ile-Ser-Gly-Leu-Gln-Thr-Glu-Asp-Glu-Ala-Asp-Tyr-Tyr-Cys-His-Ser-Tyr-Val-Ser-Ser-Phe-Asn-Val-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Thr- Val-Leu.

The variable domains, for example comprising one or more preferred CDRsas described above, preferably in the humanised form having humanantibody-derived variable framework regions, are attached to appropriateconstant domains.

The heavy and light chain constant regions can be based on antibodies ofdifferent types as desired subject to the antibody being an IgGantibody, but although they may be of or derived from those of rat ormouse origin they are preferably of or are derived from those of humanorigin. For the light chain the constant region is preferably of thelambda type and for the heavy chain it is preferably of an IgG isotype,especially IgG1, modified to effect aglycosylation as appropriate. Allhuman constant regions of the IgG isotype are known to be glycosylatedat the asparagine residue at position 297, which makes up part of theN-glycosylation motif Asparagine²⁹⁷-X²⁹⁸-Serine²⁹⁹ or Threonine²⁹⁹,where X is the residue of any amino acid except proline. The antibody ofthe invention may thus be aglycosylated by the replacement ofAsparagine²⁹⁷ in such a constant region with another amino acid whichcannot be glycosylated. Any other amino acid residue can potentially beused, but alanine is the most preferred. Alternatively, glycosylation atAsparagine²⁹⁷ can be prevented by altering one of the other residues ofthe motif, e.g. by replacing residue 298 by proline, or residue 299 byany amino acid other than serine or threonine. Techniques for performingthis site directed mutagenesis are well known to those skilled in theart and may for example be performed using a site directed mutagenesiskit such, for example, as that commercially available from Amersham. Theprocedure is further exemplified hereinafter.

It is well recognised in the art that the replacement of one amino acidin a CDR with another amino acid having similar properties, for examplethe replacement of a glutamic acid residue with an aspartic acidresidue, may not substantially alter the properties or structure of thepeptide or protein in which the substitution or substitutions were made.Thus, the aglycosylated antibodies of the present invention includethose antibodies containing the preferred CDRs but with a specifiedamino acid sequence in which such a substitution or substitutions haveoccurred without substantially altering the binding affinity andspecificity of the CDRs. Alternatively, deletions may be made in theamino acid residue sequence of the CDRs or the sequences may be extendedat one or both of the N- and C-termini whilst still retaining activity.

Preferred aglycosylated antibodies according to the present inventionare such that the affinity constant for the antigen is 10⁵ mole⁻¹ ormore, for example up to 10¹² mole⁻¹. Ligands of different affinities maybe suitable for different uses so that, for example, an affinity of 10⁶,10⁷ or 10⁸ mole⁻¹ or more may be appropriate in some cases. Howeverantibodies with an affinity in the range of 10⁶ to 10⁸ mole⁻¹ will oftenbe suitable. Conveniently the antibodies also do not exhibit anysubstantial binding affinity for other antigens. Binding affinities ofthe antibody and antibody specificity may be tested by assay proceduressuch as those described in the Examples section hereinafter, (EffectorCell Retargetting Assay), or by techniques such as ELISA and otherimmunoassays.

Antibodies according to the invention are aglycosylated IgG CD3antibodies having a “Y” shaped configuration which may have twoidentical light and two identical heavy chains and are thus bivalentwith each antigen binding site having an affinity for the CD3 antigen.Alternatively, the invention is also applicable to antibodies in whichonly one of the arms of the antibody has a binding affinity for the CD3antigen. Such antibodies may take various forms. Thus the other arm ofthe antibody may have a binding affinity for an antigen other than CD3so that the antibody is a bispecific antibody, for example as describedin U.S. Pat. No. 4,474,893 and European Patent Applications. Nos.87907123.1 and 87907124.9. Alternatively, the antibody may have only onearm which exhibits a binding affinity, such an antibody being termed“monovalent”.

Monovalent antibodies (or antibody fragments) may be prepared in anumber of ways. Glennie and Stevenson (Nature, 295, 712-713, (1982))describe a method of preparing monovalent antibodies by enzymicdigestion. Stevenson et al. describe a second approach to monovalentantibody preparation in which enzymatically produced Fab′ and Fcfragments are chemically cross-linked (Anticancer Drug Design, 3,219-230 (1989)). In these methods the resulting monovalent antibodieshave lost one of their Fab′ arms. A third method of preparing monovalentantibodies is described in European Patent No. 131424. In this approachthe “Y” shape of the antibody is maintained, but only one of the twoFab′ domains will bind to the antigen. This is achieved by introducinginto the hybridoma a gene coding for an irrelevant light chain whichwill combine with the heavy chain of the antibody to produce a mixtureof products in which the monovalent antibody is the one of interest.

More preferably, however, the monovalent aglycosylated CD3 antibodies ofthe invention are prepared by the following method. This involves theintroduction into a suitable expression system, for example a cellsystem as described hereinafter, together with genes coding for theheavy and light chains, of a gene coding for a truncated heavy chain inwhich the variable region domain and first constant region domain of theheavy chain are absent, the gene lacking the exon for each of thesedomains. This results in the production by the cell system of a mixtureof (a) antibodies which are complete bivalent antibodies, (b) antibodyfragments consisting only of two truncated heavy chains (i.e. an Fcfragment) and (c) fragments of antibody which are monovalent for the CD3antigen, consisting of a truncated heavy chain and a light chain inassociation with the normal heavy chain. Such an antibody fragment (c)is monovalent since it has any only one Fab′ arm. Production of amonovalent antibody in the form of such a fragment by this method ispreferred for a number of reasons. Thus, the resulting antibody fragmentis easy to purify from a mixture of antibodies produced by the cellsystem since, for example, it may be separable simply on the basis ofits molecular weight. This is not possible in the method of EuropeanPatent No. 131424 where the monovalent antibody produced has similarcharacteristics to a bivalent antibody in its size and outwardappearance. Additionally, the production of a monovalent antibodyfragment by the new method uses conditions which can more easily becontrolled and is thus not as haphazard as an enzyme digestion/chemicalcoupling procedure which requires the separation of a complex reactionproduct, with the additional advantage that the cell line used willcontinue to produce monovalent antibody fragments, without the need forcontinuous synthesis procedures as required in the enzymedigestion/chemical coupling procedure.

It is believed that aglycosylated antibodies according to the inventiondo not occur in nature and these aglycosylated antibodies may in generalbe produced synthetically in a number of ways. Most conveniently,however, appropriate gene constructs for the constant and variableregions of the heavy and light chains which are present in the antibodyare separately obtained and then inserted in a suitable expressionsystem.

Genes encoding the variable domains of a ligand of the desired structuremay be produced and conveniently attached to genes encoding the constantdomains of an antibody which have undergone site directed mutagenesis.These constant genes may be obtained from hybridoma cDNA or from thechromosomal DNA and have undergone mutagenesis (site directed) toproduce the aglycosylated constant regions. Genes encoding the variableregions may also be derived by gene synthesis techniques used in theidentification of the CDRs contained herein. Suitable cloning vehiclesfor the DNA may be of various types.

Expression of these genes through culture of a cell system to produce afunctional CD3 ligand is most conveniently effected by transforming asuitable prokaryotic or particularly eukaryotic cell system,particularly an immortalised mammalian cell line such as a myeloma cellline, for example the YB2/3.01/Ag20 (hereinafter referred to as Y0) ratmyeloma cell, or Chinese hamster ovary cells (although the use of plantcells is also of interest), with expression vectors which include DNAcoding for the various antibody regions, and then culturing thetransformed cell system to produce the desired antibody. Such generaltechniques of use for the manufacture of ligands according to thepresent invention are well known in the very considerable art of geneticengineering and are described in publications such as “MolecularCloning” by Sambrook, Fritsch and Maniatis, Cold Spring HarbourLaboratory Press, 1989 (2nd edition). The techniques are furtherillustrated by the Examples contained herein.

The present invention thus includes a process for the preparation of anaglycosylated IgG antibody having a binding affinity for the CD3 antigenwhich comprises culturing cells capable of expressing the antibody inorder to effect expression thereof. The invention also includes cellline which expresses an aglycosylated antibody according to theinvention.

Preferred among such cell lines are those which comprise DNA sequencesencoding the preferred CDRs described hereinbefore. A group ofnucleotide sequences coding for the CORs (a) to (f) describedhereinbefore is as indicated under (a) to (f) below, respectively, butit will be appreciated that the degeneracy of the genetic code permitsvariations to be made in these sequences whilst still encoding for theCDRs' amino acid sequences. (SEQUENCE ID NO. 17) (a) AGCTTTCCAA TGGCC(SEQUENCE ID NO. 18) (b) ACCATTAGTA CTAGTGGTGG TAGAACTTAC TATCGAGACTCCGTGAAGGG C (SEQUENCE ID NO. 19) (c) TTTCGGCAGT ACAGTGGTGG CTTTGATTAC(SEQUENCE ID NO. 20) (d) ACACTCAGCT CTGGTAACAT AGAAAACAAC TATGTGCAC(SEQUENCE ID NO. 21) (e) GATGATGATA AGAGACCGGA T (SEQUENCE ID NO. 22)(f) CATTCTTATG TTAGTAGTTT TAATGTT

Such cell lines will particularly contain larger DNA sequences whichcomprise (1) DNA expressing human heavy chain variable framework regionsand one or more of (a), (b) and (c), and (2) DNA expressing human lightchain variable framework regions and one or more of (d), (e) and (f). Aspecific example of such DNA is that sequence (l) indicated below whichcodes for the CDRs (a), (b) and (c) arranged in the heavy chainframework coded for by the human VH type III gene VH26.D.J. as discussedhereinbefore and that sequence (2) indicated below which codes for theCDRs (d), (e) and (f) arranged in the light chain framework coded for bythe human V_(L)λ type VI gene SUT. The CDR sequences (a), (b), (c), (d),(e) and (f) have been underlined. (1) (SEQUENCE ID NO. 23) GAGGTCCAACTGCTGGAGTC TGGGGGCGGT TTAGTGCAGC CTGGAGGGTC CCTGAGACTC TCCTGTGCAGCCTCAGGATT CACTTTCAGT AGCTTTCCAA TGGCCTGGGT CCGCCAGGCT CCAGGGAAGGGTCTGGAGTG GGTCTCAACCATTAGTACTA GTGGTGGTAG AACTTACTAT CGAGACTCCG TGAAGGGCCG ATTCACTATCTCCAGAGATA ATAGCAAAAA TACCCTATAC CTGCAAATGA ATAGTCTGAG GGCTGAGGACACGGCCGTCT ATTACTGTGC AAAATTTCGG CAGTACAGTG GTGGCTTTGA TTACTGGGGCCAAGGGACCC TGGTCACCGT CTCCTCA (2) (SEQUENCE ID NO. 24) GACTTCATGCTGACTCAGCC CCACTCTGTG TCTGAGTCTC CCGGAAAGAC AGTCATTATTTCTTGCACAC TCAGCTCTGG TAACATAGAA AACAACTATG TGCACTGGTA CCAGCAAAGGCCGGGAAGAG CTCCCACCAC TGTGATTTTC GATGATGATA AGAGACCGGA TGGTGTCCCTGACAGGTTCT CTGGCTCCAT TGACAGGTCT TCCAACTCAG CCTCCCTGAC AATCAGTGGTCTGCAAACTG AAGATGAAGC TGACTACTAC TGTCATTCTT ATGTTAGTAG TTTTAATGTTTTCGGCGGTG GAACAAAGCT CACTGTCCTT

The cell lines will of course also particularly contain DNA sequencesexpressing the heavy and light chain constant regions.

The humanised aglycosylated antibodies in accordance with the inventionhave therapeutic value. In particular, such aglycosylated antibodies,especially a humanised aglycosylated antibody with a specificity for thehuman CD3 antigen, has valuable applications in immunosuppression,particularly in the control of graft rejection, where it is especiallydesirable that immunosuppression is temporary rather than total, andthus that T-cells are not completely destroyed, but instead renderednon-functional by antibody blockade of the CD3 antigen—TCR complex. Inaddition, the aglycosylated CD3 antibodies may have potential in otherareas such as in the treatment of cancer, specifically in theconstruction of bispecific antibodies (for effector cell retargetting)or antibody-toxin conjugates, where the efficacy of the therapeuticagent would be compromised by Fc-mediated killing of the effector cellsor non-specific killing of Fc receptor bearing cells respectively.

In a further aspect, the invention thus includes a method of treatingpatients with cancer, particularly a lymphoma, or for immunosuppressionpurposes, for instance in a case where graft rejection may occur,comprising administering a therapeutically effective amount of anaglycosylated antibody in accordance with the invention.

Aglycosylated antibodies in accordance with the invention may beformulated for administration to patients by administering the saidantibody together with a physiologically acceptable diluent or carrier.The antibodies are preferably administered in an injectable formtogether with such a diluent or carrier which is sterile and pyrogenfree. By way of guidance it may be stated that a suitable dose ofantibody is about 1-10 mg injected daily over a time period of, forexample 10 days, although due to the elimination of the first doseresponse it will be possible if desired to adminster higher amounts ofthe antibody, for example even up to 100 mg daily, depending on theindividual patient's needs. Veterinary use is on a similar g/kg dosagebasis.

The invention is illustrated by the following Examples which areillustrated by the drawings listed below:—

FIGS. 1-8: These figures show the results of proliferation assays ofperipheral blood lymphocytes to CD3 antibodies. Four different healthyvolunteers were used. The humanised anti-lymphocyte antibody. CDw52 wasincluded as a negative control.

FIGS. 9-12: These figures show the comparison of aglycosylated CD3antibody and glycosylated CD3 antibody in a mixed lymphocyte reaction.Aglycosylated antibody specific for the mouse CD8 antigen was includedas a negative control.

FIGS. 13 & 14: These figures show the results of an Effector CellRetargetting Assay comparing glycosylated and aglycosylated IgG-type CD3antibodies. The CDw52 antibody was used as a negative control.

EXAMPLES Example 1 Preparation of an Aglycosylated Antibody Specific forthe Human CD3 Antigen Containing CDRs from the YTH 12.5 Rat Antibody inHuman Variable Framework Regions

The cloning and re-shaping of the V-region gene of the rat antibody YTH12.5 specific for the human CD3 antigen was performed as described inRoutledge et al., 1991, Eur. J. Immunol., 21, 2717 and in UK PatentApplication No. 9121126.8 and its equivalents. YTH 12.5 is a rathybridoma cell line secreting an IgG2b monoclonal antibody specific forthe CD3 antigen complex.

Briefly, the methodology was based on that of Orlandi et al., 1989, PNASUSA, 86, 3833, using the polymerase chain reaction (PCR). The VH gene(heavy chain variable region gene) was cloned using oligonucleotideprimers VHlFOR and VHLBACK. The PCR products were ligated into thevector M13-VHPCR1 in which site directed mutagenesis was performed using6 oligonucleotide primers. The V_(L) gene (light chain variable regiongene) was cloned using primers designed based on the published V_(L)λsequences. The gene was cloned into the vector M13-VKPCR, together withthe human lambda light chain constant region. In this vector mutagenesisof the V_(L) framework was performed using 5 oligonucleotides. Thehumanised V_(L) gene was then inserted into the expression vectorpHβApr-1.

A vector was generated (p316) in which the reshaped CD3 VH gene could beexpressed in conjunction with different immunoglobulin H chain constantregion genes, this vector being based on the pHβApr-gpt vector (Gunninget al., 1987, P.N.A.S. USA, 85, 7719-7723). A 1.65 Kb fragment of DNAcarrying the dihydrofolate reductase (dhft) gene and SV 40 expressionsignals (Page & Sydenham, 1991, Biotechnology, 9, 64) was inserted intothe unique EcoRI site of pHβApr-gpt. A 700 bp HindIII-BamHI DNA fragmentencoding the reshaped CD3-VH gene was then cloned into the vector'smultiple cloning site, downstream and under the control of the β actinpromoter. The desired H chain constant region gene (in genomicconfiguration) could then be inserted into the unique BamH1 restrictionenzyme site downstream of the CD3-VH gene.

The aglycosyl human IgG1 constant region was derived from the wild typeGlm (1,17) gene described by Takahashi et al., (1982, Cell, 29, 671-679)as follows. The gene was cloned into the vector M13 tg131 wheresite-directed mutagenesis was performed (Amersham International PLC) tomutate the amino acid residue at position 297 from an asparagine to analanine residue.

Oligosaccharide at Asn-297 is a characteristic feature of all normalhuman IgG antibodies (Kabat et al., 1987, Sequence of Proteins ofImmunological Interest, US Department of Health Human ServicesPublication), each of the two heavy chains in the IgG molecules having asingle branched chain carbohydrate group which is linked to the amidegroup of the asparagine residue (Rademacher and Dwek, 1984, Prog.Immunol., 5, 95-112). Substitution of asparagine with alanine preventsthe glycosylation of the antibody.

The 2.3 Kb aglycosyl IgG1 constant region was excised from M13 by doubledigestion using BamHI and BgIII and ligated into the BamHI site ofvector p316 to produce clone p323.

Subconfluent monolayers of dhfr⁻ Chinese Hamster Ovary cells wereco-transfected with the vector p323 containing the heavy chain gene anda second vector p274 containing the re-shaped human λ light chain(Routledge et al., 1991, Eur. J. Immunol., 21, 2717-2725). Prior totranfection both plasmid DNAs were linearised using the restrictionendonuclease PvuI. Transfection was carried out using the DOTMA reagent(Boehringer, Germany) following the manufacturer's recommendations.

Heavy and light chain transfectants were selected for inxanthine/hypoxanthine free IMDM containing 5% (v/v) dialysed foetal calfserum.

The production of the analogous wild type human IgG1-CD3 heavy chainvector p278 has been described elsewhere (Routledge et al., 1991, Eur.J. Immunol., 21, 2717-2725). H-chain expression vectors carrying thenon-mutant human IgG2 (Flanagan & Rabbitts, 1982, Nature 300, 709-713),IgG3 (Huck et al., 1986, Nuc. Acid. Res., 14, 1779-1789), IgG4 (Flanagan& Rabbitts, 1982, Nature 300, 709-713), Epsilon (Flanagan & Rabbitts,1982, EMBO. Journal 1, 655-660) and Alpha-2 (Flanagan & Rabbitts, 1982,Nature 300, 709-713) constant region genes (vectors p317, p318, p320,p321 and p325, respectively) were derived from the vector p316.Introduction of these vectors, in conjunction with the light chainvector p274, into dhfr⁻ CHO cells as described earlier, produced celllines secreting CD3 antibody of the γ1, γ2, γ3, γ4, ε and α-2 isotyperespectively. Cells expressing CD3 antibodies were subjected to tworounds of cloning in soft agar, and then expanded into roller bottlecultures. The immunoglobulin from approximately 4 litres of tissueculture supernatant from each cell line was concentrated by ammoniumsulphate precipitation, dialysed extensively against PBS and thenquantified as follows:

As the antibody was not pure, a competition assay was designed tospecifically quantitate the concentration of antibody with CD3 antigenbinding capacity. Human T-cell blasts were incubated with FITC labelledUCHT-1, an antibody which binds to the same epitope of the CD3 antigenas the chimaeric panel. The concentration of FITC reagent used hadpreviously been determined to be half saturating. Unlabelled YTH 12.5(HPLC purified) was titrated from a known starting concentration andadded to wells containing T-cells and UCHT-1 FITC. The unlabelledantibody serves as a competitor for the antigen binding site. This isdetected as decrease in the mean fluorescence seen when the cells arestudied using FACS analysis. Thus, titration of the chimaeric antibodiesfrom unknown starting concentrations yields a series of sigmoidal curveswhen mean fluorescence is plotted against antibody dilution. These canbe directly compared with the standard YTH 12.5 curve.

Example 2 Proliferation Assays

The capacity of a CD3 antibody to support T-cell proliferation insolution is related to the interaction of the Fc region of the antibodywith Fc receptors on accessory cells.

The aglycosylated chimaeric CD3 antibody prepared as described inExample 1 was compared with a panel of other chimaeric antibodies whichshared the same variable region architecture but different H chainconstant regions (see Example 1) for the ability to induce proliferationof human peripheral blood lymphocytes. Lymphocytes isolated from healthydonors' blood were separated on a lymphopaque gradient, washed andresuspended in IMDM containing 5% (v/v) heat-inactivated human AB serumand plated at 5×10⁴ to 1×10⁵ cells per well in plates containing CD3antibodies in solution. After 3 days in culture the cells were pulselabelled with tritiated thymidine and harvested 6 hours later and thelevel of cellular ³H incorporation was determined by scintillationcounting. The proliferation response to titrated antibody was studied infour blood donors. For a fifth donor the proliferation response wasstudied only at 1 μg. The results are shown in FIGS. 1 to 8.

For the donors studied, all ‘wild type’ antibodies led to T-cellproliferation. However the mutant, aglycosylated IgG1 isotype was nevermitogenic implicating an important role for the carbohydrate sidechains.

In a separate experiment, the ability of a panel of CD3 monoclonalantibodies in solution to stimulate T-cell mitogenesis was studied usinglymphocytes isolated from the blood of 10 donors from a variety ofethnic backgrounds. All of the naturally occurring isotypes causedproliferation in the presence of 5% human AB serum, although there wereT-cell donor dependent variations in the extent of the responses causedby the antibodies. In general, the γ1 and C monoclonal antibodies werethe most mitogenic, activating cells at the lowest concentration, andthe γ3 and γ2 were the least active. The aglycosyl derivative of the γ1monoclonal antibody was the only CD3 antibody which consistently failedto induce T-cell proliferation in any of the donors tested, givingresponses equivalent to those of the non-activating control monoclonalantibody Campath-1H. In order to exclude endotoxin contamination as thecause of the proliferation seen with the α2 and ε preparations, it wasconfirmed that proliferation could be blocked by the addition of anexcess of the aglycosyl CD3 mAb thus implicating the CD3 antigen in theactivation process.

The total lack of proliferative response seen with the aglycosyl γ1 CD3monoclonal antibody was surprising, given its position in the ECRactivity hierarchy (see Example 5 below). The reason for this inactivityis probably due to its reduced affinity for FcRs rather than becauseaglycosylation has abolished the ability to trigger some post-bindingevent required for proliferation, e.g. by destroying a secondaryrecognition site on the monoclonal antibody. This is supported by theobservation that the aglycosyl γ1 mAb could stimulate proliferation to aconsiderable degree if the assay was performed in IgG-free medium. Thereis probably a minimum thereshold level for the number of contacts orstrength of interaction between T-cell and accessory cell which must beexceeded before proliferation can be initiated, and this cannot beachieved by the aglycosyl γ1 mAb in the presence of significant levelsof competing immunoglobulin.

Example 3 The Effect of Chimaeric CD3 Antibodies in Mixed LymphocyteReactions

A series of experiments was conducted to test whether the aglycosylatedantibody IgGlAg of Example 1 had the capacity to block T-cellproliferation in a mixed lymphocyte reaction (MLR) and the results of 2experiments are shown in FIGS. 9 to 12.

Peripheral blood lymphocytes were isolated from two blood donors. Thestimulator cell population was caesium irradiated. The responderpopulation was incubated with titrated antibody for 30 minutes beforethe irradiated stimulator cells were added (FIGS. 9, 10 and 11). Controlwells of responder cells were also incubated with irradiated respondercells at each antibody condition, to determine the specific effect ofthe antibody on the responder cells (FIGS. 9, 10 and 12).

After 5 days incubation the wells were pulse labelled with tritiatedthymidine and harvested 6 hours later.

The aglycosylated antibody does block the mixed lymphocyte reaction; asthe antibody is titrated out the blockade effect is less and theproliferation increases. The ‘wild type’ IgG1 actually has a mitogeniceffect on the T-cells and so any blockade of the MLR is not seen throughthis response.

An ‘irrelevant’ aglycosylated antibody specific for the murine CD8antigen was included as a negative control. This antibody has asexpected no effect on the MLR.

Example 4 In Vivo Effect of IgG1 Antibodies

In vivo experiments were performed with chimaeric anti-human CD3antibodies in mice which were transgenic for the human CD3 epsilonsubunit including the aglycosylated antibody of Example 1 (IgGlAg). Theability of some of the chimaeric CD3 antibodies to cause the release ofTNF factor following a single injection was compared.

In this set of experiments serum was collected from the mice beforeinjection and then at 90 minutes and 4 hours following intravenousinjection with 10 μg of relevant CD3 antibody. Groups consisted of 5mice and the serum collected from each group was pooled. Analysis of thelevel of TNF in the serum was performed in the laboratory of ProfessorJean-Francois Bach, using a bioassay which measured the cytotoxic effectof sera on the L929 mouse fibroblast cells as a result of the presenceof TNF.

The results are shown in Table 1 below: TABLE 1 TNF detected in serum ofhCD3 mice following injection with chimaeric CD3 antibodies Prebleed 90minute 4 hour Sample TNF level TNF level TNF level Saline 0 units/ml  0units/ml 0 units/ml IgG1 0 units/ml >400 units/ml    0 units/ml IgG2 0units/ml >400 units/ml    0 units/ml IgG1Ag 0 units/ml 50 units/ml 0units/ml IgE 0 units/ml 50 units/ml 25 units/ml  YTH 12.5 0 units/ml 50units/ml 0 units/ml

A significant difference is seen between the level of TNF associatedwith injection of the two forms of human IgG1. The aglycosylated form isassociated with at least an eight-fold less release of TNF than the wildtype IgG1, or with the IgG2 antibody. The results of Examples 2-4 showthat the aglycosylated CD3 antibody was not mitogenic to T-cells insolution indicating that the antibody had a reduced capacity to interactwith Fc receptors on accessory cells. The antibody retained theimmunosuppressive properties that are characteristic of CD3 antibodies.In vivo the aglycosylated antibody led to a significantly lower releaseof tumour necrosis factor in human CD3 transgenic mice than the parentalIgG1 antibody. Thus this agent may be an ‘improved’ CD3 antibody for thepurposes of immunosuppression if the decreased TNF release seen in miceis mirrored in humans.

Example 5 Effector Cell Retargetting Assays for the Detection of CD3Antibodies with the Ability to Direct T-Cell Killing

This was performed as described elsewhere (Gilliland et al., 1988, PNASUSA, 85, 4419) and measures the ability of a CD3 monoclonal antibody tocross-link activated T-cells to FcγR bearing target cells and thus tomediate target cell lysis. Briefly U937 human monocytic human cellswhich express the Fcγ receptors I, II and III were labelled with ⁵Crsodium chromate and resuspended to 2×10⁵ cells per ml. These cells wereused as targets. Human T cell blasts, generated from human peripheralblood lymphocytes by activation with mitogenic CD3 antibody followed byculture in medium containing IL-2, were used as the effector cells. Theywere washed and resuspended at a concentration of 2×10⁵ cells ml⁻¹ priorto use in the assay. 100 μl volumes of the purified chimaeric antibodypreparations were diluted in 3-fold steps in the wells of a microtitreplate. 50 μl each of the effector and target cells were then added toeach well and the mixture was incubated at 37° C. for at least 4 hours.After this time 100 μl of supernatant was removed and assayed forreleased ⁵¹Cr. Each antibody dilution was tested in duplicate.

The U937 monocytic cell line expresses human F-c receptors and can belysed by activated human T-cell blasts in the presence of CD3 monoclonalantibodies capable of cross-linking the two cell types. The results(FIGS. 13 and 14) show that when aglycosylated, the human IgG1 antibodyof Example 1 is still able to cross-link 1-cells to the U937 cells,albeit at a reduced level, and thus redirect T-cell cytotoxicity. Thiswas a surprising finding since, given the published data, the effectivekilling mediated by aglycosyl γ1 monoclonal antibody was unexpected.

There existed the possibility that removal of the carbohydrate had madethis monoclonal antibody especially sticky, and so able to bind to U937cells without interacting with FcγRs. However, it was subsequentlydemonstrated (results not shown) that the ability of the aglycosyl γ1monoclonal antibody to mediate the destruction of mouse L cell targets(a mouse cell line which expresses the human FcγRI) was dependent on theexpression of a transfected human FcγRI gene, thus confirming the FcγRbinding activity of this monoclonal antibody. We conclude that the ECRassay is a particularly sensitive method of detecting Fc-FcRinteractions.

The ECR results indicate that the hierarchy of binding of the IgGchimaeric antibodies is γ2<γ3<Agγ1<γ4<γ1. If the assumption is made thatthe mitogenic activity of an antibody is predicted by its Fc receptorbinding ability, then one would expect the above hierarchy to bedisplayed in the T cell proliferation assays. However, this was not thecase; the order of activities in T cell proliferation experiments (1 to3) was Agγ1<γ2<γ4<γ3<γ1. This demonstrates that the mitogenicity of anantibody cannot be predicted in a straightforward fashion from theresults of assays which measure Fc-Fc receptor interactions. This viewis supported by the behaviour of the epsilon chimaeric antibody whichperformed poorly in the ECR assay and yet consistently had the highestmitogenic activity. This suggests that antibodies can activate T cellsby binding to something other than Fcγ receptors (as displayed on U937cells) on accessory cells, i.e. an inability to bind to Fcγ receptors isno guarantee that an antibody will not be mitogenic.

1-27. (canceled)
 28. A method of treating a patient having cancer whichcomprises administering to said patient a therapeutically effectiveamount of a ligand having a binding affinity for the CD3 antigencomplex.